Project Summary Project 2 ? Microbially-liberated urea nitrogen and host essential amino acids. The elderly are particularly susceptible to sarcopenic obesity (SAO), whereby a loss of lean mass (e.g., muscle) accompanies an increase in fat, and which is difficult to treat via life style changes (e.g., diet) designed to target obesity. Although numerous studies have addressed the role of diet and gut microbes as factors in diseases such as obesity, the potential beneficial role of gut microbes in preventing loss of lean mass in disorders such as SAO has received comparably less attention. Urea-nitrogen salvage (UNS) is the process whereby urea is degraded by gut microbes releasing nitrogen that can be utilized by gut microbes to produce amino acids, or be reabsorbed and used by the host. UNS likely plays an important role in nitrogen metabolism in humans, and evidence suggests both UNS and the production of essential amino acids (EAAs) by gut microbes may have important impacts on human health. Yet, despite the availability of microbially-liberated urea-nitrogen (MLUN) for EAA synthesis in the gut, these two microbial components of host nitrogen metabolism have been studied primarily independently and with little focus on the structure and function of the gut microbial community. The goal of this project is to determine the gut microbiota potential for use of MLUN in synthesis of EAAs, and demonstrate the provision of microbially-synthesized EAAs for host protein synthesis. We use the arctic ground squirrel (AGS) as a study species, as AGS are able to preserve lean mass during their long hibernation season. Our hypothesis is that MLUN in the gut of AGS is utilized by gut microbes for synthesis of EAAs which are incorporated into host tissues during hibernation. Our approach relies upon simultaneous analyses of the structure and function of the gut microbial community, microbial production of EAAs using MLUN, and incorporation of microbial-derived EAAs in host tissues under varying conditions of host dietary protein availability and physiological state. In Specific Aims 1 and 2 we utilize isotopically labeled urea (13C and 15N), which when injected intra-peritoneally diffuses into the gut and becomes available for urea degrading microbes. After injecting hibernating squirrels with labeled urea, we will measure 13CO2 in breath (to confirm ureolytic activity), determine the EAA synthesis potential of the gut microbiota using next generation sequencing techniques (metagenomics and metatranscriptomics to look for EAA biosynthesis genes) and metabolomics (15N-NMR to search for labeled EAAs in gut contents), and determine if AGS incorporate microbially-derived EAAs into host tissues (15N-NMR). In Specific Aim 3, we will isolate and characterize bacteria from the GI tract of AGS and determine culture conditions necessary for growth to experiment with mock gut communities in the future. Our approach promises to yield an increased knowledge of the role of the gut microbial community in host nitrogen metabolism and protein conservation, contributing to our understanding of the relationship between the gut microbial community and human health.